Polynucleotide encoding a human chemotactic protein

ABSTRACT

A human chemotactic protein polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for preventing and/or treating for stem cell mobilization, myeloprotection and neuronal protection, to treat tumors, to promote wound healing, to combat parasitic infection and to regulate hematopoiesis. Also disclosed are antagonists against such polypeptides which may be employed to treat rheumatoid arthritis, lung inflammation, allergy, infectious diseases and to prevent inflammation and atherosclerosis. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention for detecting diseases are also disclosed.

This application is a continuation-in-part of application Ser. No.08/424,425 filed in the United States Patent and Trademark Office onApr. 21, 1995, abandoned, and pending application Ser. No.PCT/US94/05384 filed under the Patent Cooperation Treaty on May 16,1994.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is a chemotactic protein. The invention also relates toinhibiting the action of such polypeptides.

RELATED ART

There are three forms of monocyte chemotactic protein, namely, MCP-1,MCP-2 and MCP-3. All of these proteins have been structurally andfunctionally characterized and have also been cloned and expressed.MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes, andleukocytes), while MCP-3 also attracts eosinophils and T lymphocytes(Dahinderi, E. et al., J. Exp. Med., 179:751-756 (1994)).

Initially, human monocyte-specific attracting factor, was purified froma glioma cell line and a monocytic cell line. Matsushima, K. et al, J.Exp. Med., 169:1485-1490 (1989). This factor was originally designatedglioma-derived chemotactic factor (GDCF) and monocyte chemotactic andactivating factor (MCAF) by Matsushima, et al. This factor is nowreferred to as MCP-1. Subsequent cloning of the cDNA for MCP-1 showed itto be highly similar to the murine JE gene. The JE gene could bemassively induced in murine fibroblasts by platelet-derived growthfactor. Cochran, B. H., et al, Cell 33:939-947 (1983). Murine JE ishighly similar to MCP-1. The MCP-1 protein is 62% identical to murine JEin a region of 68 shared N-terminal residues. It is widely accepted thatJE and MCP-1 are species homologs.

A method of suppressing tumor formation in a vertebrate by administeringJE/MCP-1 has been disclosed in PCT application WO-92/20372, along withmethods of treating localized complications of malignancies and methodsof combatting parasitic infection by administering JE/MCP-1. Expressionof the JE/MCP-1 protein in malignant cells was found to suppress thecells ability to form tumors in vivo.

Human MCP-1 is a basic peptide of 76 amino acids with a predictedmolecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly inmonocytes, endothelial cells and fibroblasts. Leonard, E. J. andYoshimura, T., Immunol. Today, 11:97-101 (1990). The factors whichinduce this expression is IL-1, TNF or lipopolysaccharide treatment.

Other properties of MCP-1 include the ability to strongly activatemature human basophils in a pertussis toxin-sensitive manner. MCP-1 is acytokine capable of directly inducing histamine release by basophils,(Bischoff, S. C. et al., J. Exp. Med., 175:1271-1275 (1992)).Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophilspretreated with Interleukin 3, Interleukin 5, or granulocyte/macrophagecolony-stimulating factor. MCP-1 induced basophil mediator release mayplay an important role in allergic inflammation and other pathologiesexpressing MCP-1.

Clones having a nucleotide sequence encoding a human monocytechemotactic and activating factor (MCAF) reveal the primary structure ofthe MCAF polypeptide to be composed of a putative signal peptidesequence of 23 amino acid residues and a mature MCAF sequence of 76amino acid residues. Furutani, Y. H., et al, Biochem. Biophys. Res.Commu., 159:249-55 (1989). The complete amino acid sequence of humanglioma-derived monocyte chemotactic factor (GDCF-2) has also beendetermined. This peptide attracts human monocytes but not neutrophils.It was established that GDCF-2 comprises 76 amino acid residues. Thepeptide chain contains 4 half-cysteines, at positions 11, 12, 36 and 52,which create a pair of loops, clustered at the disulfide bridges.Further, the MCP-1 gene has been designated to human chromosome 17.Mehrabian, M. R., et al, Genomics, 9:200-3 (1991). Certain data suggeststhat a potential role for MCP-1 is mediating monocytic infiltration ofthe artery wall. Monocytes appear to be central to atherogenesis both asthe progenitors of foam cells and as a potential source of growthfactors mediating intimal hyperplasia. Nelken, N. A., et al, J. Clin.Invest., 88:1121-7 (1991). It has also been found that synovialproduction of MCP-1 may play an important role in the recruitment ofmononuclear phagocytes during inflammation associated with rheumatoidarthritis and that synovial tissue macrophages are the dominant sourceof this cytokine. MCP-1 levels were found to be significantly higher insynovial fluid from rheumatoid arthritis patients compared to synovialfluid from osteoarthritis patients or from patients with otherarthritides. Koch, A. E., et al, J. Clin. Invest., 90:772-9 (1992).

MCP-2 and MCP-3 are classified in a subfamily of proinflammatoryproteins and are functionally related to MCP-1 because they specificallyattract monocytes, but not neutrophils. Van Damme, J., et al, J. Exp.Med., 176:59-65 (1992). MCP-3 shows 71% and 58% amino acid homology toMCP-1 and MCP-2 respectively. MCP-3 is an inflammatory cytokine thatregulates macrophage functions.

The transplantation of hemolymphopoietic stem cells has been proposed inthe treatment of cancer and hematological disorders. May studiesdemonstrate that transplantation of hematopoietic stem cells harvestedfrom the peripheral blood has advantages over the transplantation ofmarrow-derived stem cells. Due to the low number of circulating stemcells, there is a need for induction of pluripotent marrow stem cellmobilization into the peripheral blood. Reducing the amount of blood tobe processed to obtain an adequate amount of stem cells would increasethe use of autotransplantation procedures and eliminate the risk ofgraph versus host reaction connected with allotransplantation.Presently, blood mobilization of marrow CD34⁺ stem cells is obtained bythe injection of a combination of agents, including antiblastic drugsand G-CSF or GM-CSF. Drugs which are capable of stem cell mobilizationinclude IL-1, IL-7, IL-8, and NIP-1α. Both IL-1 and IL-8 demonstrateproinflammatory activity that may be dangerous for good engrafting. IL-7must be administered at high doses over a long duration and MIP-1α isnot very active as a single agent and shows best activity when incombination with G-CSF.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptide of the present invention is ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding a polypeptide of thepresent invention including mRNAs, DNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, forstem cell mobilization, myeloprotection and neuronal protection, totreat tumors, to promote wound healing, to combat parasitic infectionand to regulate hematopoiesis.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with another aspect of the present invention, there areprovided agonists which mimic the polypeptide of the present inventionand bind to receptors to elicit second messenger responses.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof rheumatoid arthritis, lung inflammation, allergy, infectious diseasesand to prevent inflammation and atherosclerosis.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in the nucleic acid sequences encodinga polypeptide of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, for example, synthesis of DNA andmanufacture of DNA vectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 depicts the cDNA sequence (SEQ ID NO:1) and corresponding deducedamino acid sequence (SEQ ID NO:2) of the chemotactic protein of thepresent invention (CP). The 119 amino acid sequence shown is the fulllength protein, with approximately the first 26 amino acids representinga leader sequence (underlined) such that the mature form of the proteinis 93 amino acids in length. The standard one letter abbreviation foramino acids is used.

FIG. 2 illustrates a comparison of the amino acid sequence homologybetween the polypeptide of the present invention (SEQ ID NO:2), MCP-1(SEQ ID NO:5) and MIP-1α (SEQ ID NO:6). The polypeptide of the presentinvention (CP) shows 39% homology with MIP-1α and 34% homology withMCP-1.

FIG. 3 illustrates the chemotactic activity of the polypeptide of thepresent invention on neutrophils (PMN) and peripheral blood mononuclearcells (PBMC). Neutrophils and peripheral blood mononuclear cells wereisolated from peripheral blood, loaded with calcein-AM and used forchemocaxis in a 96 well, single-use Neuroprobe chemotactic chamber.After 90 minutes incubation with the polypeptide of the presentinvention (CP), the chamber was dismounted, the filter air-dried and thenumber of cells which migrated through the membrane quantitated in acytofluor II.

FIG. 4 illustrates that the polypeptide of the present invention (CP)inhibits the growth and differentiation of high proliferative potentialcolony forming cells (HPP-CFC) (A) and is not effective on lowproliferative potential colony forming cells (LPP-CFC) (B). In theseexperiments, 1,500 cells from low density, non-adherent bone marrowcells were plated in agar-medium supplemented with 5 ng/ml mouse IL-3,100 ng/ml mouse SCF, 10 ng/ml mouse IL-1α, 5 ng/ml human M-CSF, and withor without the indicated concentrations of the polypeptide of thepresent invention (CP). Colonies were scored after 14 days ofincubation. Three experiments were performed. The results are presentedas mean number of colonies±SD. An irrelevant protein had no effects.

FIG. 5 shows the effect of the polypeptide of the present invention (CP)on bone marrow cells which were enriched in the primitive Lin-cells byremoving committed precursor cells (antibodies anti-CD11b, CD4, CD8,CD45R and Gr.-1). The panel A shows ratios±SD of LPP-CFC/HPP-CFC in thebone marrow cells (column 1) or Lin-cells (column 2) plated inagar-medium with 5 ng/ml IL-3, 100 ng/ml SCF, 10 ng/ml IL-1a, 5 ng/mlM-CSF. Columns 3, 4 and 5 show the ratio of LPP-CFC/HPP-CFC found in theLin-cells that were cultured with 5 ng/ml IL-3 and 100 ng/ml SCF (column3), IL-3, SCF and 50 ng/ml of the polypeptide of the present invention(CP) (column 4) or IL-3, SCF and 50 ng/ml of an irrelevant protein(column 5). After 6 days, cultures were assayed for HPP-CFC and LPP-CFC.The panel B shows the cellularity after 6 days incubation.

FIG. 6 illustrates that the polypeptide of the present invention (CP)protects HPP-CFC but not LPP-CFC from the cytotoxic effect of cytosinearabinoside (Ara-C) in vitro.

FIG. 7 illustrates that, the polypeptide of the present invention (CP)protects HPP-CFC but not LPP-CFC from the cytotoxic effect of5-Fluorouracil (5-FU) in vitro.

FIG. 8 illustrates the effect of the polypeptide of the presentinvention (CP) and Basic FGF on Cortical Neuronal Survival.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited at the American Type Culture Collection, 12301 Park LawnDrive, Rockville Md. 20852, as ATCC Deposit No. 75703 on Mar. 10, 1994.

The polynucleotide of this invention was discovered from an activatedmonocyte cDNA library. It contains an open reading frame encoding aprotein of approximately 119 amino acids in length of which the first 26amino residues comprise a putative leader sequence. The mature proteintherefore is predicted to be 93 amino acids in length. It isstructurally related to mouse monocyte chemotactic protein (MCP-1 orJE), showing 27% identity, and 56% similarity over the entire humanMCP-1 protein sequence. The polypeptide contains all four cysteineresidues that occur in all chemokines in a characteristic motif. Thespacing between these cysteines is conserved compared with the murineMCP-1/JE which strongly suggests that the new gene is a chemokine.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 (SEQ IDNO:1) or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) or for the mature polypeptide encoded by the depositedcDNA may include, but is not limited to: only the coding sequence forthe mature polypeptide; the coding sequence for the mature polypeptideand additional coding sequence such as a leader or secretory sequence ora proprotein sequence; the coding sequence for the mature polypeptide(and optionally additional coding sequence) and non-coding sequence,such as introns or non-coding sequence 5' and/or 3' of the codingsequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which has theamino acid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 "backbone" sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The polypeptide of the present invention (CP), may be employed for thepromotion of wound healing. Since CP is a chemokine, it is achemo-attractant for leukocytes (such as basophils, PMNs, PBLs etc.);therefore, it causes infiltration of target immune cells to a woundarea.

The polypeptide of the present invention (CP) may also be employed as ananti-tumor treatment and for treating localized complications of amalignancy, such as pleural effusions or ascites.

The presence of MCPs in vivo is accompanied by a local increase in thepresence of eosinophils which have the distinctive function of killingthe larvae of parasites that invade tissues, as in schistosomiasis,trichinosis and ascariasis. Therefore, the polypeptide of the presentinvention (CP) may be employed for combatting parasitic infections.

The polypeptide of the present invention may be employed for mobilizinghematopoietic progenitor cells into the peripheral blood circulation ofa non-human and human host, preferably a human host, for subsequentrecovery and use thereof in transplantation. The polypeptide of thepresent invention is administered in an amount effective to mobilizeinto and increase the amount of hematopoietic progenitor cells in theperipheral blood, in particular, increase the amount of humanhematopoietic stem cells in the peripheral blood. Such cells are oftenreferred to as CD34+ cells. For example, the polypeptide is administeredin amounts as hereinafter described. The polypeptide of the presentinvention may be administered alone or in conjunction with other agents,for example, GM-CSF and G-CSF which are known to be effective forincreasing such cells in peripheral blood. Mobilization of hematopoieticprogenitor cells into the peripheral circulation is important forautologous and heterologous bone marrow transfers which are used, forexample for treatment of cancer and hematological disorders.

The polypeptide of the present invention may also be employed to inhibitdestruction of hematopoietic progenitor cells in a non-human and humanhost, preferably a human host, resulting from treatment withchemotherapeutic agents. The polypeptide of the present invention may beadministered prior to, during or subsequent to chemotherapy and allows ahigher dose of chemotherapy to be employed in the treatment of cancer.The polypeptide of the present invention is administered in an amounteffective to inhibit destruction of hematopoietic progenitor cells; forexample, the polypeptide is administered in amounts as hereinafterdescribed. The polypeptide may be administered alone or in conjunctionwith other agents. The polypeptide of the present invention may also beemployed to protect hematopoietic progenitor cells to thereby prevent orinhibit diseases which may result from the destruction thereof; forexample, leukopenia, myelodysplastic syndrome, and neutropenia.

The polypeptide of the present invention may also be employed in amountseffective to inhibit the degeneration of neuronal cells in non-human andhuman hosts, preferably a human host, which results from neuronaldegenerative diseases such as Alzheimer's disease, Parkinson's diseaseand AIDS-related complex. For example, the polypeptide may be employedin amounts as hereinafter described.

                                      TABLE 1    __________________________________________________________________________    Effect of CP administration to mice on the distribution of the primitive    hematopoietic progenitors in peripheral blood, spleen, and bone marrow    after two days           Numbers of Progenitors per           10.sup.4 PB cells   10.sup.4 Spleen cells                                                  10.sup.4 BM cells    Treatment           HPP   LPP    IM     HPP    LPP   IM    HPP   LPP    __________________________________________________________________________    Saline 0.5 ± 0.7                 38 ± 9.5                        6.5 ± 1.9                               0.7 ± 1.5                                      5.5 ± 2.5                                            1.5 ± 2.3                                                  53 ± 11                                                        484 ± 59    CP     3.5 ± 0.5                  95 ± 16.9                          25 ± 13.5                               2.75 ± 0.9                                      4.2 ± 3.5                                            3.5 ± 2.4                                                   27 ± 3.5                                                        610 ± 28    (1 mg/kg/day)    __________________________________________________________________________     PB = Peripheral blood, mononuclear cells     Spl. = Low density fraction of spleen cells     BM = Bone marrow fraction that is 6fold enriched for the primitive cells     HPP = High proliferative potential colony forming cells     LPP = Low proliferative potential colony forming cells     IM = Immature cell, a rare cell type found in the bone marrow, gives rise     to a highly refrectile, small (<50 cells/colony) colony in the presence o     multiple cytokines; the cells within the colony are stacked in a     horizontal plane and they exhibit blast cell like nuclear staining     characteristics.     Three mice were injected IP daily with either the polypeptide of the     present invention (CP) or saline. Forty eight hours after the first     injection, blood was collected from each animal by cardiac puncture and     mice were then sacrificed to obtain bone marrow and spleen. Indicated     numbers of cells from each of the tissues were then plated in duplicates     in agarcontaining medium in the presence of rmIL3 (5 ng/ml), rmSCF (50     ng/ml), rhMCSF (5 ng/ml), and rmIL1a (10 ng/ml) and  incubated for 14     days. Data are pooled from three animals in each group and expressed as     mean ± S.D.

                                      TABLE 2    __________________________________________________________________________    Effect of CP administration to mice on the distribution of the primitive    hematopoietic progenitors in peripheral blood, spleen, and bone marrow    after four days            Numbers of Progenitors per            10.sup.4 PB cells     10.sup.4 Spleen cells                                                       10.sup.4 BM cells    Treatment            HPP   LPP     IM      HPP    LPP     IM    HPP   LPP    __________________________________________________________________________    Saline  0     29 ± 5.6                          1 ± 1.5                                  1 ± 0.6                                         10 ± 4.6                                                 0.8 ± 0.7                                                       60 ± 8                                                             505 ± 45    CP      3.8 ± 1.5                  84.5 ± 14.5                          28.6 ± 8.6                                  2.6 ± 0.5                                         10.3 ± 2.1                                                   7 ± 1.5                                                       26.5                                                             330 ± 46    (1 mg/kg/day)    __________________________________________________________________________     PB = Peripheral blood, mononuclear cells     Spl. = Low density fraction of spleen cells     BM = Bone marrow fraction that is 6fold enriched for the primitive cells     HPP = High proliferative potential colony forming cells     LPP = Low proliferative potential colony forming cells     IM = Immature cell, a rare cell type found in the bone marrow, gives rise     to a highly refrectile, small (<50 cells/colony) colony in the presence o     multiple cytokines; the cells within the colony are stacked in a     horizontal plane and they exhibit blast cell like nuclear staining     characteristics.     Three mice were injected IP daily with either the polypeptide of the     present invention (CP) or saline. Ninety six hours after the first     injection, blood was collected from each animal by cardiac puncture and     mice were then sacrificed to obtain bone marrow and spleen. Indicated     numbers of cells from each of the tissues were then plated in duplicates     in agarcontaining medium in the presence of rmIL3 (5 ng/ml), rmSCF (50     ng/ml), rhMCSF (5 ng/ml), and rmIL1a (10 ng/ml) and  incubated for 14     days. Data are pooled from three animals in each group and expressed as     mean ± S.D.

                  TABLE 3    ______________________________________    Analysis of the peripheral blood leukocyte composition by    FACSan in mice administered with CP after two days    Percent Positive in the Gated the Cell Populations           CD45R +  GR.1 +    Mac. 1 +                                      CD8 +  CD4 +    Treatment           B-Cells  PMN       Monocytes                                      T-cells                                             T-cells    ______________________________________    Saline 40.5 ± 9.2                    62.5 ± 10.6                              19.5 ± 2.1                                      29 ± 5.6                                             39 ± 12    CP       37 ± 5.6                      56 ± 11.3                                18 ± 4.2                                      27 ± 4.3                                             33 ± 7    (mg/Kg/    day)    ______________________________________     Three C57 Black 6 mice (˜20 g weight) were injected (IP) daily with     either saline or the polypeptide of the present invention (CP). Forty     eight hours after the first injection, blood was collected by cardiac     puncture and mice were sacrificed to obtain spleen and bone marrow cells.     For immunostaining, 0.1 ml of blood from each of the animal was first     treated with Gen Trak lysing solution to lyse the red blood cells.     Nucleated cells were then sedimented, washed with PBS, and incubated  wit     PEconjugated monoclonal antibodies against CD45R, Gr. 1, Mac. 1, CD4, &     CD8 and processed for flowcytometry. At least 10,000 cells were analyzed.     Data are expressed as mean percent positive cells in the appropriate     channels ± SD.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

This invention provides a method for identification of the receptor forthe polypeptide of the present invention (CP). The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to CP. Transfected cells which are grown on glassslides are exposed to labeled the polypeptide of the present invention(CP). The polypeptide of the present invention (CP) can be labeled by avariety of means including iodination or inclusion of a recognition sitefor a site-specific protein kinase. Following fixation and incubation,the slides are subjected to auto-radiographic analysis. Positive poolsare identified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor. As an alternativeapproach for receptor identification, labeled ligand can bephotoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

This invention also provides a method of screening compounds to identifyagonists and antagonists to the polypeptide of the present invention. Asan example, a mammalian cell or membrane preparation expressing a CPreceptor would be contacted with a compound of interest. The ability ofthe compound to generate a the response of a known second messengersystem following interaction with the CP receptor is then measured. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis. The ability of acompound to bind the CP receptor and elicit a second messenger responseidentifies that compound as an agonist. A compound which binds but doesnot elicit a second messenger response identifies that compound as anantagonist.

A competitive binding assay, in which the compounds are labeled, forexample by radioactivity may also be employed to identify antagonists.Such methods are known in the art.

Antagonists include negative dominant mutants of the polypeptide of thepresent invention (CP). The polypeptide of the present invention (CP) isa tetrameric polypeptide wherein one mutated unit will cause the entirepolypeptide to be non-functional. A negative dominant mutant of thepolypeptide of the present invention (CP) binds to the CP receptor butfails to activate cells (leukocytes) to which it binds. An assay todetect negative dominant mutants of the polypeptide of the presentinvention (CP) is an in vitro chemotaxis assay wherein a multiwellchemotaxis chamber equipped with polyvinylpyrrolidone-free polycarbonatemembranes is used to measure the chemoattractant ability of thepolypeptide of the present invention (CP) for leukocytes in the presenceand absence of potential antagonist or agonist molecules.

Potential antagonists also include an antibody, or in some cases, anoligopeptide, which binds to the polypeptide and prevents it frombinding its receptor.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5' coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix--see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of the polypeptide of thepresent invention (CP). The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of the mRNA molecule into thepolypeptide of the present invention (CP) (Antisense--Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptide of the present invention (CP).

Potential antagonists include a small molecule which binds to andoccupies the active site of the polypeptide thereby making the catalyticsite inaccessible to substrate such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

The antagonists may be employed to treat inflammation by preventing theattraction of monocytes to a wound or a site of trauma, and to regulatenormal pulmonary macrophage populations, since acute and chronicinflammatory pulmonary diseases are associated with sequestration ofmononuclear phagocytes in the lung.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinabove described.

The polypeptides, and agonists and antagonists, of the present inventionmay be employed in combination with a suitable pharmaceutical carrier.Such compositions comprise a therapeutically effective amount of thepolypeptide or agonist or antagonist, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, or agonists and antagonists, of the present invention maybe employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, parenterally, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

The polypeptides and agonists and antagonists which are polypeptides mayalso be employed in accordance with the present invention by expressionof such polypeptides in vivo, which is often referred to as "genetherapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described) ; the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X VT-19-17-H2, ψCRE, ψCRIP, GP+E-86 GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of the polypeptide of the present invention(CP).

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding thepolypeptide of the present invention (CP) can be used to identify andanalyze mutations. For example, deletions and insertions can be detectedby a change in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA or alternatively, radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, U.S.A., 85:4397-4401(1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of the polypeptide of the present invention in varioustissues since an over-expression of the proteins compared to normalcontrol tissue samples can detect the presence of the polypeptide of thepresent invention (CP). Assays used to detect levels of the polypeptideof the present invention in a sample derived from a host are well-knownto those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis and preferably anELISA assay. An ELISA assay initially comprises preparing an antibodyspecific to the CP antigen, preferably a monoclonal antibody. Inaddition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or in this example a horseradishperoxidase enzyme. A sample is now removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attached to any of the polypeptide of thepresent invention attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound to thepolypeptide of the present invention. Unattached reporter antibody isthen washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of the polypeptide of the present invention present in agiven volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention (CP) are attached to a solidsupport and labeled CP and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of the polypeptide of thepresent invention in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLES Example 1

Bacterial Expression and Purification of CP

The DNA sequence encoding for the polypeptide of the present inventionCP, ATCC # 75703, is initially amplified using PCR oligonucleotideprimers corresponding to the 5' and 3' sequences of the processed CPprotein (minus the signal peptide sequence) and the vector sequences 3'to the CP gene. Additional nucleotides corresponding to CP were added tothe 5' and 3' sequences respectively. The 5' oligonucleotide primer hasthe sequence 5' TCAGGATCCCCTACGGGCTCGTGGTC 3' (SEQ ID NO:3) contains aBam H1 restriction enzyme site followed by 18 nucleotides of CP codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3' sequence 3' CGCTCTAGAGTAAAACGACGGCCAGT 5' (SEQ IDNO:4) contains complementary sequences to the XbaI site and to apBluescript SK⁻ vector sequence located 3' to the CP DNA insert. Therestriction enzyme sites correspond to the restriction enzyme sites onthe bacterial expression vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with Bam H1 and Xba I.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain m15/rep4available from Qiagen under the trademark M15/rep 4 by the proceduredescribed in Sambrook, J. et al, Molecular Cloning: A Laboratory Manual,Cold Spring Laboratory Press, 1989. M15/rep4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG("Isopropyl-B-D-thiogalacto pyranoside") was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized polypeptide of the presentinvention (CP) was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography411:177-184 (1984). The polypeptide of the present invention (CP) (95%pure) was eluted from the column in 6 molar guanidine HCl pH 5.0 and forthe purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mMsodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolarglutathione (oxidized). After incubation in this solution for 12 hoursthe protein was dialyzed to 10 mmolar sodium phosphate.

Example 2

Expression pattern of CP in human cells

Northern blot analysis was carried out to examine the levels ofexpression of the polypeptide of the present invention (CP) in humancells. Total cellular RNA samples were isolated with RNAzol™ B system(Biotecx Laboratories, Inc. 6023 South Loop East, Houston, Tex. 77033).About 10 μg of total RNA isolated from each human tissue specified wasseparated on 1% agarose gel and blotted onto a nylon filter. (Sambrook,Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press,(1989)). The labeling reaction was done according to the StratagenePrime-It kit with 50 ng DNA fragment. The labeled DNA was purified witha Select-G-50 column. (5 Prime--3 Prime, Inc. 5603 Arapahoe Road,Boulder, Colo. 80303). The filter was then hybridized with radioactivelabeled full length CP gene at 1,000,000 cpm/ml in 0.5M NaPO₄, pH 7.4and 7% SDS overnight at 65° C. After wash twice at room temperature andtwice at 60° C. with 0.5×SSC, 0.1% SDS, the filter was then exposed at-70° C. overnight with an intensifying screen. The message RNA for CP isabundant in activated and unactivated T cells, monocytes and T celllines.

Example 3

Cloning and expression of CP using the baculovirus expression system

The DNA sequence encoding the full length polypeptide of the presentinvention (CP) protein, ATCC # 75703, Is amplified using PCRoligonucleotide primers corresponding to the 5' and 3' sequences of thegene:

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit ("Geneclean," BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with restriction endonucleasescorresponding to the amplified products and then purified again on a 1%agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the polypeptide of the present invention (CP)protein using the baculovirus expression system (for review see:Summers, M. D. and Smith, G. E. 1987, A manual of methods forbaculovirus vectors and insect cell culture procedures, TexasAgricultural Experimental Station Bulletin No. 1555). This expressionvector contains the strong polyhedrin promoter of the Autographacalifornica nuclear polyhedrosis virus (AcMNPV) followed by therecognition sites for the restriction endonucleases used to digest theamplified products. The polyadenylation site of the simian virus (SV)40is used for efficient polyadenylation. For an easy selection ofrecombinant virus the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA was then isolated from a 1% agarose gel using thecommercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E. coli HB101 cells are then transformed and bacteria identifiedthat contained the plasmid (pBacCP) with the CP gene using the enzymes.The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBacCP is co-transfected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacCP are mixedin a sterile well of a microtiter plate containing 50 μg of serum freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards10 μl Lipofectin plus 90 μl Grace's medium are added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with "Blue Gal" (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a "plaque assay" can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus is added to the cells,blue stained plaques are picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses is then resuspended in anEppendorf tube containing 200 μl of Grace's medium. The agar is removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-CP at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Example 4

Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer contains an EcoRI site and the 3' primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 5

Primary Indication of CP as a Mobilizer of Marrow Stem Cells (BoneMarrow Rescue)

The effect of the polypeptide of the present invention (CP) on thedistribution of the primitive hematopoietic progenitors in peripheralblood, spleen, and bone marrow was studied in 16 week old C57B1/6 mice(about 20 g). In the first experiment, 3 mice were injected i.p. dailywith 1 mg/kg of the polypeptide of the present invention (CP) or salinefor 2 days and analyzed 24 hours after the last injection. In the secondexperiment, another 3 mice were injected i.p. daily with 1 mg/kg of thepolypeptide of the present invention (CP) or saline for 4 days andanalyzed 24 hours after the last injection. In both the experiments, theblood of each animal was collected by cardiac puncture and the mice weresacrificed to obtain bone marrow and spleens. The indicated number ofcells from each of the tissues was then plated in duplicates inagar-containing medium in the presence of 5 ng/ml IL-3, 50 ng/ml SCF, 5ng/ml M-CSF and 10 ng/ml IL-1α and incubated for 14 days. In the 2experiments, the data from the different animals were pooled andexpressed as mean±S.D. The results of both experiments shows that thepolypeptide of the present invention (CP) mobilize stem cells from bonemarrow to peripheral blood Tables 1 and 2!. In the first experiment,after 2 days of treatment with the polypeptide of the present invention(CP), the frequency of HPP-CFC, LPP-CFC and immature cells in peripheralblood increased significantly over the controls. No changes wereobserved in the spleen and a significant decrement of HPP-CFC wasobserved in the bone marrow Table 1!. In the second experiment, after 4days of treatment with the polypeptide of the present invention (CP),the same significant increment of HPP-CFC, LPP-CFC and immature cellsfrequency was observed in peripheral blood. A significant increment ofimmature cells frequency was observed in the spleen and a significantdecrement of HPP-CFC and LPP-CFC was observed in the bone marrow Table2!. In particular it is important to note the presence of immaturehematopoietic cells in the peripheral blood after the injection of thepolypeptide of the present invention (CP). The effect was observed inthe animals treated with the polypeptide of the present invention (CP)was not due to toxicity as the FACScan profile of the leukocytecomposition of both the control and the mice treated with CP isidentical Table 3!.

Example 6

CP as a Myeloprotectant against cytosine arabinoside

In this experiment, Lin-cells were plated (1×10⁵ cell/ml) in a growthmedium that was supplemented with 5 ng/ml mouse IL-3, 50 ng/ml mouse SCF(column 1); IL-3, SCF and 100 ng/ml of the polypeptide of the presentinvention (CP) (column 2); or IL-3, SCF and 100 ng/ml of the irrelevantprotein HG200-3-B (column 3). AFter 48 hours of incubation, one set ofthe above cultures received 50 μg/ml Ara-C and the incubation was thencontinued for an additional 24 hours. Cells were then harvested, washedthree times with HBSS to remove the drug and the cytokines, and assayedfor the presence of HPP-CFC and LPP-CFC as described in the legend toFIG. 4. The results are expressed as mean % of protection (±SD). The %of protection was calculated as follows: Percent protection is expressedas number of colonies found in cultures incubated in the presence ofAra-C divided by the number of colonies found in cultures incubatedwithout Ara-C×100. Data from one out of 3 experiments are shown in FIG.6. All the samples were tested in duplicates.

Example 7

CP as a Myeloprotectant against 5-Fluorouracil

Mononuclear population of mouse bone marrow cells was depleted oflineage-committed cells by negative selection using a panel monoclonalantibodies directed against cell surface antigens. The resultingpopulation of cells (Lin.-cells) were resuspended (1×10⁵ cells/ml) in agrowth medium containing IL-3 (5 ng/ml), SCF (50 ng/ml), GM-CSF (5ng/ml), M-CSF (5 ng/ml) and IL-1α (10 ng/ml) and 1 ml of this cellsuspension was dispensed into culture tubes. (1) A set of duplicatecultures received no chemokine; (2) duplicate cultures with thepolypeptide of the present invention (CP) at 100 ng/ml; and (3)duplicate cultures with an irrelevant protein at 100 ng/ml. All cultureswere incubated in a tissue culture incubator for 48 hours, at whichpoint one culture from each set received 5-Fluorouracil at 100 μg/ml andincubation was continued for additional 24 hours. All cultures were thenharvested, washed three times with HBSS, and then assayed for thepresence of the HPP-CFC & LPP-CFC as described in the legend to FIGS. 5Aand 5B. Percent protection is expressed as number of colonies detectedin cultures incubated in the presence of 5-FU divided by the number ofcolonies found in cultures incubated without 5-FU×100. Data areexpressed as Mean±SD. Two experiments were performed and each assay wasin duplicates. See FIG. 7.

Example 8

CP effect on cortical neuronal survival

Sprague-Dawley rats at gestation day 17 were sacrificed and the cortexwas removed and the meninges were carefully pealed away from thecortical tissue pieces. Single cell suspensions were prepared and thecells were plated in medium containing 5% horse serum at a density of20,000 cells/well. After 24 hours the serum containing medium wasremoved and serum-free medium was added to the cultures. Included in theserum-free cultures was a concentration of the polypeptide of thepresent invention (CP) as shown in FIG. 8. The polypeptide of thepresent invention (CP) is a CP polypeptide encoded by the polynucleotidesequence as shown in SEQ ID NO:1 of the application. The medium waschanged every other day and the polypeptide of the present invention(CP) was added again. The neurons were maintained in culture for 6 daysprior to the viability assay.

Cell viability was assessed using the live/dead assay kit from MolecularProbes. This assay is a two-color fluorescence cell viability assaybased on the simultaneous determination of live and dead cells. Livecells are distinguished by the presence of ubiquitous intracellularesterase activity, determined by enzymatic conversion of the nearlynon-fluorescent cell permeant calcein AM to the intensely fluorescentcalcein. The polycationic calcein is well retained by living cells andthus produces an intense uniform green fluorescence in living cells.Thus the emission reading (approximately 530 nm) is a measurement of thetotal cell number of the cultures. As shown in FIG. 8, the number oflive cells increased as the concentration of the polypeptide of thepresent invention (CP) increased.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 6    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 360 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..357    (ix) FEATURE:    (A) NAME/KEY: sig.sub.-- peptide    (B) LOCATION: 1..79    (ix) FEATURE:    (A) NAME/KEY: mat.sub.-- peptide    (B) LOCATION: 79..357    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGGCAGGCCTGATGACCATAGTAACCAGCCTTCTGTTCCTTGGTGTC48    MetAlaGlyLeuMetThrIleValThrSerLeuLeuPheLeuGlyVal    26-25-20-15    TGTGCCCACCACATCATCCCTACGGGCTCTGTGGTCATACCCTCTCCC96    CysAlaHisHisIleIleProThrGlySerValValIleProSerPro    10-515    TGCTGCATGTTCTTTGTTTCCAAGAGAATTCCTGAGAACCGAGTGGTC144    CysCysMetPhePheValSerLysArgIleProGluAsnArgValVal    101520    AGCTACCAGCTGTCCAGCAGGAGCACATGCCTCAAGGCAGGAGTGATC192    SerTyrGlnLeuSerSerArgSerThrCysLeuLysAlaGlyValIle    253035    TTCACCACCAAGAAGGGCCAGCAGTTCTGTGGCGACCCCAAGCAGGAG240    PheThrThrLysLysGlyGlnGlnPheCysGlyAspProLysGlnGlu    404550    TGGGTCCAGAGGTACATGAAGAACCTGGACGCCAAGCAGAAGAAGGCT288    TrpValGlnArgTyrMetLysAsnLeuAspAlaLysGlnLysLysAla    55606570    TCCCCTAGGGCCAGGGCAGTGGCTGTCAAGGGCCCTGTCCAGAGATAT336    SerProArgAlaArgAlaValAlaValLysGlyProValGlnArgTyr    758085    CCTGGCAACCAAACCACCTGCTAA360    ProGlyAsnGlnThrThrCys    90    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 119 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaGlyLeuMetThrIleValThrSerLeuLeuPheLeuGlyVal    26-25-20-15    CysAlaHisHisIleIleProThrGlySerValValIleProSerPro    10-515    CysCysMetPhePheValSerLysArgIleProGluAsnArgValVal    101520    SerTyrGlnLeuSerSerArgSerThrCysLeuLysAlaGlyValIle    253035    PheThrThrLysLysGlyGlnGlnPheCysGlyAspProLysGlnGlu    404550    TrpValGlnArgTyrMetLysAsnLeuAspAlaLysGlnLysLysAla    55606570    SerProArgAlaArgAlaValAlaValLysGlyProValGlnArgTyr    758085    ProGlyAsnGlnThrThrCys    90    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TCAGGATCCCCTACGGGCTCGTGGTC26    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    TGACCGGCAGCAAAATGAGATCTCGC26    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 99 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetLysValSerAlaAlaLeuLeuCysLeuLeuLeuIleAlaAlaThr    151015    PheIleProGlnGlyLeuAlaGlnProAspAlaIleAsnAlaProVal    202530    ThrCysCysTyrAsnPheThrAsnArgLysIleSerValGlnArgLeu    354045    AlaSerTyrArgArgIleThrSerSerLysCysProLysGluAlaVal    505560    IlePheLysThrIleValAlaLysGluIleCysAlaAspProLysGln    65707580    LysTrpValGlnAspSerMetAspHisLeuAspLysGlnThrGlnThr    859095    ProLysThr    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 93 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetGlnValSerThrAlaAlaLeuAlaValLeuLeuCysThrMetAla    151015    LeuCysAsnGlnValLeuSerAlaProLeuAlaAlaAspThrProThr    202530    AlaCysCysPheSerTyrThrSerArgGlnIleProGlnAsnPheIle    354045    AlaAspTyrPheGluThrSerSerGlnCysSerLysProSerValIle    505560    PheLeuThrLysArgGlyArgGlnValCysAlaAspProSerGluGlu    65707580    TrpValGlnLysTyrValSerAspLeuGluLeuSerAla    8590    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide that hybridizes underthe stringent conditions of 0.5M NaPO₄, 7% SDS at 65° C. sequence to amember selected from the group consisting of:(a) a polynucleotideencoding a polypeptide comprising amino acid -26 to 93 of SEQ ID NO. 2;(b) a polynucleotide encoding a polypeptide comprising amino acid -25 to93 of SEQ ID NO. 2; (c) a polynucleotide encoding a polypeptidecomprising amino acid 1 to 93 of SEQ ID NO. 2; and (d) the complement of(a), (b), or (c).
 2. The isolated polynucleotide of claim 1 wherein saidmember is (a).
 3. The isolated polynucleotide of claim 1 wherein saidmember is (b).
 4. The isolated polynucleotide of claim 1 wherein saidmember is (c).
 5. The isolated polynucleotide of claim 1, wherein thepolynucleotide is DNA.
 6. The isolated polynucleotide of claim 1,wherein the polynucleotide is RNA.
 7. A method of making a recombinantvector comprising inserting the isolated polynucleotide of claim 1 intoa vector, wherein said polynucleotide is DNA.
 8. A recombinant vectorcomprising the polynucleotide of claim 1, wherein said polynucleotide isDNA.
 9. A recombinant host cell comprising the polynucleotide of claim1, wherein said polynucleotide is DNA.
 10. The isolated polynucleotideof claim 1 comprising nucleotides 79 to 360 of SEQ ID NO. 1 or adegenerate variant thereof.
 11. The isolated polynucleotide of claim 10comprising nucleotides 79 to 360 of SEQ ID NO.
 1. 12. The isolatedpolynucleotide of claim 1 comprising the polynucleotide of SEQ ID NO. 1or a degenerate variant thereof.
 13. The isolated polynucleotide ofclaim 12 comprising the polynucleotide of SEQ ID NO.
 1. 14. An isolatedpolynucleotide that hybridizes under the stringent conditions of 0.5MNaPO₄, 7% SDS at 65° C. sequence to a member selected from the groupconsisting of:(a) a polynucleotide encoding the same polypeptide encodedby the human cDNA in ATCC Deposit No. 75703; (b) a polynucleotideencoding the same mature polypeptide encoded by the human cDNA in ATCCDeposit No. 75703; and (c) the complement of (a) or (b).
 15. Theisolated polynucleotide of claim 14, wherein the member is (a).
 16. Theisolated polynucleotide of claim 14, wherein the member is (b).
 17. Theisolated polynucleotide of claim 14 wherein said polynucleotidecomprises DNA identical to the coding portion of the human cDNA in ATCCDeposit No. 75703 which encodes for a mature polypeptide.
 18. A methodfor producing a polypeptide comprising the steps of:(I) expressing in arecombinant host cell the polypeptide encoded by a polynucleotideselected from the group consisting of:(a) a polynucleotide encoding apolypeptide comprising amino acid -26 to 93 of SEQ ID NO. 2; (b) apolynucleotide encoding a polypeptide comprising amino acid -25 to 93 ofSEQ ID NO. 2; and (c) a polynucleotide encoding a polypeptide comprisingamino acid 1 to 93 of SEQ ID NO. 2; and (II) recovering the expressedpolypeptide.
 19. The method of claim 18, wherein said polynucleotide is(a).
 20. The method of claim 18, wherein said polynucleotide is (b). 21.The method of claim 18, wherein said polynucleotide is (c).
 22. Anisolated polynucleotide encoding a polypeptide wherein, except for atleast one conservative amino acid substitution, said polypeptide has anamino acid sequence that is identical to a member selected from thegroup consisting of:(a) amino acids -26 to 93 of SEQ ID NO:2; (b) aminoacids -25 to 93 of SEQ ID NO:2; and (c) amino acids 1 to 93 of SEQ IDNO:2 wherein forming cells (HPP-CFC) in vitro; orsaid polypeptide, inmature form, exhibits at least one of the following two activities; (1)inhabits growth of differentiations of high proliferative potentialcolony forming cells (HPP-CFC) in vitro; or (2) increases the frequencyof hematopoietic progenitor cells in peripheral blood when injectedintraperitoneally or intravenously into a mouse.
 23. The isolatedpolynucleotide of claim 22, wherein said member is (a).
 24. The isolatedpolynucleotide of claim 22, wherein said member is (b).
 25. The isolatedpolynucleotide of claim 22, wherein said member is (c).